Three-dimensional imaging using swept, confocally aligned planar excitation and sample perturbation

Abstract

In some embodiments, a SCAPE system routes light from a tilted intermediate image plane to an infinity space disposed behind a third objective. A first beam splitter positioned in the infinity space routes light from the intermediate image plane with different wavelengths in different directions. First and second light detector arrays capture first and second wavelength images, respectively, and optical components route light having the first and second wavelength towards the first and second light detectors, respectively. In some embodiments, a SCAPE system is used to capture a plurality of images while a sample is perturbed (e.g., vibrated, deformed, pushed, pulled, stretched, or squeezed) in order to visualize the impact of the perturbation on the sample.

What is claimed is: 
     
       1. An imaging apparatus comprising:
 a first set of optical components having a proximal end and a distal end, wherein the first set of optical components includes a first objective disposed at the distal end of the first set of optical components; 
 a second set of optical components having a proximal end and a distal end, wherein the second set of optical components includes a second objective disposed at the distal end of the second set of optical components; 
 a scanning element that is disposed proximally with respect to the proximal end of the first set of optical components and proximally with respect to the proximal end of the second set of optical components,
 wherein the scanning element is arranged to route a sheet of excitation light so that the sheet of excitation light will pass through the first set of optical components in a proximal to distal direction and project into a sample that is positioned distally beyond the distal end of the first set of optical components, wherein the sheet of excitation light is projected into the sample at an oblique angle, and wherein the sheet of excitation light is projected into the sample at a position that varies depending on an orientation of the scanning element, 
 wherein the first set of optical components routes detection light from the sample in a distal to proximal direction back to the scanning element, and 
 wherein the scanning element is also arranged to route the detection light so that the detection light will pass through the second set of optical components in a proximal to distal direction and form an intermediate image plane at a position that is distally beyond the distal end of the second set of optical components; 
 
 an actuator configured to perturb the sample while the sheet of excitation light is projected into the sample at a plurality of positions that correspond, respectively, to a plurality of different orientations of the scanning element; 
 a light detector array arranged to capture a plurality of images of the intermediate image plane that correspond, respectively, to the plurality of different orientations of the scanning element; and 
 a processor configured to, based on the captured images, quantify displacement or speed of motion of features in the sample and provide a measure of at least one mechanical property. 
 
     
     
       2. The imaging apparatus of  claim 1 , wherein the actuator comprises a vibrator. 
     
     
       3. The imaging apparatus of  claim 1 , wherein the actuator comprises at least one of an audio frequency transducer and an ultrasound frequency transducer. 
     
     
       4. The imaging apparatus of  claim 1 , wherein the actuator is configured to deform, push, pull, stretch, or squeeze the sample. 
     
     
       5. A method of imaging a sample comprising:
 projecting a sheet of excitation light into a sample at an oblique angle, wherein a position of the sheet of excitation light within the sample varies depending on an orientation of a scanning element; 
 forming, from detection light that originates from the position of the sheet of excitation light, an image at an intermediate image plane, wherein the intermediate image plane remains stationary regardless of the orientation of the scanning element; 
 perturbing the sample while the sheet of excitation light is projected into the sample at a plurality of positions that correspond, respectively, to a plurality of different orientations of the scanning element; 
 capturing a plurality of images of the intermediate image plane that correspond, respectively, to the plurality of different orientations of the scanning element; and 
 quantifying, based on the captured images, displacement or speed of motion of features in the sample and providing a measure of at least one mechanical property. 
 
     
     
       6. The method of  claim 5 , wherein the perturbing comprises vibrating. 
     
     
       7. The method of  claim 5 , wherein the perturbing comprises perturbing at an audio frequency or an ultrasound frequency. 
     
     
       8. The method of  claim 5 , wherein the perturbing comprises at least one of pushing, pulling, stretching, or squeezing the sample. 
     
     
       9. An imaging apparatus comprising:
 a light source; 
 at least one optical component that generates a sheet of light from the light source; 
 a beam splitter disposed in a path of the sheet of light; 
 a scanning mirror disposed in a path of the sheet of light; 
 a first telescope having a proximal end and a distal end, and a first objective disposed at the distal end of the first telescope; 
 a second telescope having a proximal end and a distal end, and a second objective disposed at the distal end of the second telescope, 
 wherein the beam splitter routes the sheet of light towards the scanning mirror, wherein the scanning mirror routes the sheet of light into the proximal end of the first telescope, wherein the first telescope routes the sheet of light in a proximal to distal direction through the first objective into a sample, accepts return light from the sample through the first objective and routes the return light in a distal to proximal direction back to the scanning mirror, wherein the scanning mirror routes the return light through the beam splitter and into the proximal end of the second telescope, and wherein the second telescope routes the sheet of light in a proximal to distal direction through the second objective to form a tilted intermediate image plane; 
 a camera optically positioned to capture images at the tilted intermediate image plane; 
 an actuator configured to perturb the sample; and 
 
       a processor configured to, based on the captured images, quantify displacement or speed of motion of features in the sample and provide a measure of at least one mechanical property. 
     
     
       10. The imaging apparatus of  claim 9 , wherein the actuator comprises a vibrator. 
     
     
       11. The imaging apparatus of  claim 9 , wherein the actuator comprises at least one of an audio frequency transducer and an ultrasound frequency transducer. 
     
     
       12. The imaging apparatus of  claim 9 , wherein the actuator is configured to deform, push, pull, stretch, or squeeze the sample.